Low selectivity index of ivermectin and macrocyclic lactones on SARS-CoV2 replication in vitro argues against their therapeutic use for COVID-19

  1. Christine Chable-Bessia
  2. Charlotte Boullé
  3. Aymeric Neyret
  4. Jitandrya Swain
  5. Mathilde Hénaut
  6. Peggy Merida
  7. Nathalie Gros
  8. Alain Makinson
  9. Sébastien Lyonnais
  10. Cédric B. Chesnais
  11. Delphine Muriaux

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Abstract

There are very limited antiviral therapeutic options for coronavirus infections, therefore global drug re-purposing efforts are paramount to identify available compounds that could provide clinical benefits to patients with COVID-19. Ivermectin was first approved for human use as an endectocide in the 1980s. It remains one of the most important global health medicines in history and has recently been shown to exert in vitro activity against SARS-CoV-2. However, the macrocyclic lactone family of compounds has not previously been evaluated for activity against SARS-CoV-2. The present study aims at comparing their anti-viral activity in relevant pulmonary cell lines in vitro. Here, in vitro antiviral activity of the avermectins (ivermectin and selamectin) and milbemycins (moxidectin and milbemycin oxime) were assessed against a clinical isolate from a CHU Montpellier patient infected with SARS-CoV-2 in 2020. Ivermectin demonstrated anti-SARS-CoV-2 activity in vitro in human pulmonary cells in comparison to VeroE6 (with EC 50 of 1-3 μM). Similarly, the other macrocyclic lactones moxidectin, milbemycin oxime and selamectin reduced SARS-CoV-2 replication in vitro (with EC 50 of 2-5 μM). Immunofluorescence assays with ivermectin and moxidectin showed a reduction in the number of infected and polynuclear cells suggesting a drug action on viral cell fusion. However, cellular toxicity of the avermectins and milbemycins during infection showed a very low selectivity index <10 for all compounds. In conclusion, none of these agents appears suitable for human use for its anti-SARS-CoV-2 activity per se , due to low selectivity index. This is discussed in regards to recent clinical COVID studies on ivermectin.

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  1. ScreenIT

    SciScore for 10.1101/2021.11.01.466865: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Incubation with primary antibodies anti-SARS-CoV-2 rabbit membrane (M) protein (Rockland) (1:100) was performed for 2 hours at room temperature.
    primary antibodies anti-SARS-CoV-2 rabbit membrane (M) protein (Rockland)
    suggested: None
    anti-SARS-CoV-2 rabbit membrane (M) protein (Rockland)
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Antiviral activity was assessed by cytopathic effect (CPE) reduction assay on Vero E6 cells or by RT-qPCR on A549-hACE2 or Calu-3 cells.
    Vero E6
    suggested: None
    Calu-3
    suggested: BCRJ Cat# 0264, RRID:CVCL_0609)
    Quantitative reverse transcription polymerase chain reaction (RT-qPCR): Viral particles were collected from virus-infected A549 hACE2 cell supernatants at 72 hours post infection (100 μL) and viral RNAs were isolated from the supernatants using the Quick RNA Viral 96 Kit (Zymo Research) according to the manufacturer’s instructions.
    A549
    suggested: None
    Immunofluorescence and confocal microscopy infected cell imaging: A549-hACE2 cells seeded on glass coverslips were infected with SARS-CoV-2 at a MOI 0.01.
    A549-hACE2
    suggested: RRID:CVCL_A5KB)
    Software and Algorithms
    SentencesResources
    The 50% effective concentration (EC50 [the concentration required to inhibit virus-induced cell death by 50%]) and the 50% cytotoxic concentration (CC50) (the concentration that reduces the viability of uninfected cells to 50% of that of untreated control cells) were determined using 4-parameter nonlinear regression with GraphPad Prism v8.0, based on the following calculations: Data are mean ± sd of three biological replicates, each of which consisted of duplicate samples.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    DAPI-stained nuclei surfaces were analyzed using ZEN software (Otsu threshold method) from a grid of 8×8 images taken at 20x magnification.
    ZEN
    suggested: None
    For intensity analysis, cells were imaged as Z stack with 0.3 μm sections then Z projection images were processed for total intensity per cell using ImageJ/Fiji.
    ImageJ/Fiji
    suggested: None
    Statistical tests were performed using Origin 8.5 software.
    Origin
    suggested: (Origin, RRID:SCR_014212)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Limitations of this study: Our study is the first to examine antiviral parameters of macrocyclic lactones other than IVM on SARS-CoV-2, nevertheless the data we provide here are only in vitro data, and the recent developments encourage us to emphasize that they should therefore be taken cautiously, and that many steps are to be completed before any result can be translated into clinical practice. Clinical risk-benefit balance is not in favor of the use of any of these molecules at this early stage. In order to try to approach the pharmacokinetic parameters of the molecules concerned, we used the data available for avermectins and milbemycins, often obtained from animal data; which do not directly allow an extrapolation to humans. Nevertheless, the concordance of all the data suggesting the impossibility of reaching even an imperfect pharmacological target of Cmax:EC50 and therefore even less able to reach Cmin:EC90 seemed important to us to avoid inappropriate conclusions being reached drawn from these data. Next steps and conclusion: Altogether our results clearly show that none of these agents is suitable for human use for its anti-SARS-CoV-2 activity per se. Our data show that DOR, MIL, and SEL are not clinically relevant candidates. They are not approved for human use, and therefore should not be used, and there is no rationale in our data to conduct the extensive studies that this would require (toxicology, impurity profile, thorough ADME evaluation). Further steps could...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 32. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  2. Version published to 10.1101/2021.11.01.466865v1 on bioRxiv